The present invention relates to therapeutic methods utilizing a botulinum neurotoxin such as for treating cardiac risk factors (e.g. hypertension, diabetes, hyperlipidemia, and obesity) and/or respiratory disorders (e.g. asthma, bronchitis and chronic obstructive pulmonary disease (COPD)) and/or arthritis. More particularly, the present invention relates to methods for treating various cardiac risk factors and/or respiratory disorders and/or arthritis utilizing local administration of at least one botulinum neurotoxin.
Coronary Risk Factors
A coronary (or cardiac) risk factor is a condition and/or behavior that increases a patient's chances of developing a coronary heart disease. A coronary heart disease is also called coronary artery disease (CAD), ischaemic heart disease or atherosclerotic heart disease and is the end result of the accumulation of atheromatous plaques in walls of the arteries that supply heart muscle with oxygen and nutrients. The fewer total number of risk factors that a patient has, the less risk the patient has of developing a coronary heart disease. Additionally, the greater the level of a particular risk factor (i.e. a clinically measurable aspect of the risk factor, for example having a total blood cholesterol level of 200 mg/dL or greater rather than below 200 mg/dL), the greater is the risk that the patient will develop a coronary heart disease.
Some coronary risk factors cannot be controlled. Examples of uncontrollable coronary risk factors include, for example, age and genetic disposition. The risk of developing some coronary heart disease simply increases with every passing year. For example men ages 45 and older and women ages 55 and older are at increased risk of developing coronary heart disease as compared to younger persons. Another factor to consider is family history. If a person is the child of parents who developed coronary heart disease before the age of 55, such offspring are more likely to develop coronary heart disease themselves than their peers whose parents developed coronary heart disease after the age 55 or not at all. Lastly, studies have shown a person's racial or ethnic background can also be considered a risk factor for developing coronary heart disease, where African Americans, Mexican Americans, American Indians, and other Native Americans are at greater risk than Caucasians.
Some coronary risk factors can be controlled. Examples of controllable coronary risk factors include physical inactivity, smoking, being overweight or obese, hypertension (high blood pressure), high blood cholesterol and having diabetes. People with inactive lifestyles simply have an increased risk of developing heart disease at some point in their life. In order to reduce this risk, it is generally advised that a person participate in 30-60 minutes of physical activity on most days. People who smoke cigarettes have the greatest risk among the general population of smokers (smoking being a risk factor in and of itself, as it interferes with the ability of the body to prevent blood clotting), with those who smoke cigars or pipes having a risk of developing coronary heart disease that is less than those that smoke cigarettes. Even if one does not smoke, exposure to other people's second-hand smoke increases the risk of developing cardiovascular disease. Naturally then, it follows that quitting smoking helps to reduce the risk of developing and suffering coronary heart disease.
Being overweight and/or obese is also a coronary risk factor for developing coronary heart disease. Persons having too much body fat are at an increased risk for developing coronary heart disease and/or eventually experiencing a cardiac event, including instant death or a nonfatal infarction. In particular, women with waist measurements of more than 35 inches and men with waist measurements of more than 40 inches (having too much fat around the waist), increases that person's risk of developing heart disease. Another method to measure if a person is at risk is to determine their Body Mass Index (BMI). A BMI number is a number calculated and based upon a person's weight and height. For most people, the BMI number is a reliable indicator of the amount of fat the person carries, and is typically used by health care professionals to screen for weight categories that may lead to health problems, such as diabetes and coronary heart disease. Persons having a BMI value of 25 or greater are considered to be at the highest risk of developing coronary heart disease.
Hypertension, or high blood pressure, is blood pressure of about 140/90 mmHg or higher. Nearly 1 in 3 American adults has high blood pressure. Unfortunately, many people that suffer from high blood pressure are unaware they have elevated pressures until they experience trouble with their heart, brain, or kidneys. If not treated, hypertension can lead to heart enlargement, aneurysms in blood vessels such as at the aorta and arteries in the brain, legs, and intestines. Furthermore, hypertension can lead to blood vessel narrowing in the kidney, which may cause a kidney to fail. Additionally and as stated above, hypertension is one of the many coronary risk factors, and can lead to hardening of the arteries in the body, especially those in the heart, brain and kidneys which can lead to a heart attack, a stroke, or kidney failure.
Having high blood cholesterol and/or high triglyceride levels are additional coronary risk factors. The term hyperlipidemia means high lipid levels, and while hyperlipidemia includes several conditions, it usually means that a patient has high cholesterol and high triglyceride levels. Persons having total blood cholesterol level of 200 mg/dL (milligrams/per deciliter) or higher and/or triglyceride levels above 150 mm/dL have increased risk for developing coronary heart disease. People that already have other risk factors and have low-density lipoprotein (LDL) cholesterol levels of 100 mg/dL or higher are at increased risk also. Persons with no other risk factors but having low-density lipoprotein (LDL) levels of 160 mg/dL or higher, and/or with high-density lipoprotein (HDL) cholesterol levels of less than 40 mg/dL, are also considered to have an increased risk of developing coronary heart disease. Commonly prescribed statins (or HMG-CoA reductase inhibitors) are a class of drugs that are used to lower cholesterol levels in people with or at risk of cardiovascular disease. Cholesterol is lowered by inhibition of HMG-CoA reductase, which is the rate-limiting enzyme of the pathway of cholesterol synthesis, which stimulates LDL receptors, resulting in an increased clearance of low-density lipoprotein (LDL) from the bloodstream and a decrease in blood cholesterol levels. Additionally, maintaining a “heart-healthy” diet and increased exercise is also advised to patients having high blood cholesterol and/or high triglyceride levels.
Diabetes is another coronary risk factor. Diabetes mellitus is a chronic disease in which blood glucose (sugar) levels are too high. Normal regulation of the hormone, insulin, is responsible for maintaining proper glucose levels in the blood. Abnormally high levels of glucose can damage the small and large blood vessels, leading to diabetic blindness, kidney disease, amputations of limbs, stroke, and heart disease. Generally, there are two types of diabetes, Type 1 diabetes is usually (but not always) diagnosed in children and young adults. The islets of Langerhans, in the pancreas of people who have type 1 diabetes, do not produce insulin, and thus such people rely on external insulin, typically injected subcutaneously or as recently developed inhaled. People with type 2 diabetes mellitus have insulin resistance, not enough insulin (low insulin production), or both; they may or may not eventually require externally supplied insulin to control their glucose levels and can take oral, systemic medication such as, metformin (FORTAMET, GLUCOPHAGE, and RIOMET). About 17 million people in America have Diabetes mellitus, and about 1 million new cases are diagnosed each year.
Respiratory Disorders
It has been estimated that about 350,000 people in the United States die from lung disease and that lung disease is the number three killer in America, responsible for one in seven deaths. About 25 million Americans live with chronic lung disease, which affect people of all ages and genders.
Bronchitis, asthma and chronic obstructive pulmonary disease (COPD) are example of some respiratory disorders.
Chronic obstructive pulmonary disease (COPD) is a chronic lung disease, marked by damage to the lungs and includes two main illnesses: chronic bronchitis and emphysema, both of which make breathing difficult. In COPD, the respiratory airways and air sacs (alveoli) lose their shape, become slack and in some cases, the walls between sacs are even destroyed. Additionally, excessive mucus is produced in the airways, as well as the walls of the airways become inflamed and thickened. As a result, less air gets in and less air goes out of the lungs. Unfortunately, there is no cure for COPD.
Cigarette smoking is the most common cause of COPD, and breathing other kinds of lung irritants such as pollution, dust, or chemical fumes over a long period of time may also cause or contribute to COPD.
Bronchitis is an inflammation of the bronchi (medium-size airways) in the lungs which can be acute (e.g. caused by a virus, bacteria, dust and fumes) or chronic. In persons with chronic bronchitis, the bronchial tubes become permanently thickened and/or inflamed. The patient with chronic bronchitis typically exhibits a persistent, continuous cough with mucus. A person is diagnosed as having chronic bronchitis if they cough most days for at least three months a year in two consecutive years. Smoking, air pollution and dust or toxic gases can contribute to the chronic bronchitis. In some instances, chronic inflammation of the airways may lead to asthma. Typical treatment includes antibiotics (if bacterial), rest, ingestion of copious amounts of fluids, and over-the-counter cough medication.
Asthma is typically an allergic disorder of respiration, characterized by bronchospasm, wheezing, and difficulty in expiration. It can also be accompanied by coughing and feelings of chest constriction. Asthma occurs when the main bronchial tubes are inflamed, resulting in a tightening of the muscles of the bronchial walls, and can be accompanied by excessive mucus production. As a result, wheezing up to and including severe difficulty in breathing can be brought on. In some instances the severity of the constriction is such that the person experiences an asthma attack, which can be life-threatening.
The signs of asthma and symptoms can vary from person to person and from episode to episode and can range from mild to severe. Occasional asthma episodes with mild, short-lived symptoms such as wheezing can be experienced wherein between episodes no difficulty in breathing is experienced. Other asthma sufferers may experience chronic coughing and wheezing punctuated by severe asthma attacks, which are typically preceded by warming signs, such as increased shortness of breath or wheezing, coughing, chest tightness or pain. In children, an audible whistling or wheezing sound when exhaling can sometimes be heard, even without a stethoscope (especially after vigorous activities e.g. running, playing, climbing etc. . . . ) and frequent coughing spasms.
Medications to treat asthma vary from person to person. In general, a combination of long-term control medications and quick relief medications is typically utilized. Medications generally fall into one of three categories: long-term-control medications, quick-relief medications and medications for allergy-induced asthma. Long-term control medications are usually taken every day on a long-term basis, to control persistent asthma, while quick relief medications are utilized to relieve symptoms of short-term, asthma attacks. For allergy-induced asthma, medications are taken to decrease a person's sensitivity to a particular allergen and prevent or decrease an immune system reaction to a particular allergen or allergens.
Exemplary long term medications to treat asthma include inhaled corticosteroids which are anti-inflammatory drugs that reduce inflammation in the airways and prevent blood vessels from leaking fluid into the airway tissues. Exemplary inhaled corticosteroids include fluticasone (FLOVENT), budesonide (PULMICORT), triamcinolone (AZMACORT), flunisolide (AEROBID) and beclomethasone (QVAR). Another long-term medication are the long-acting beta-2 agonists (LABAs), bronchodilators that dialate constricted airways. Examples include salmeterol (SEREVENT DISKUS) and formoterol (FORADIL). Still additional long term medications include leukotriene modifiers, which reduce the production or block the action of leukotrienes, which are release by cells in the lungs during an asthma attack. Leukotrienes release results in inflamed airways, leading to wheezing, mucus overproduction and coughing. Exemplary leukotriene modifiers include montelukast (SINGLULAIR) and zafirlukast (ACCOLATE).
Additional long term medications to treat asthma include cromolyn (INTAL) and nedocromil (TILADE), which require daily inhaled use, to help prevent attacks of mild to moderate asthma. Theophylline (dimethylxanthine) requires daily administration, which is a bronchodilator in pill form.
Quick-relief medications are typically short active bronchodilators which are designed to address the symptoms of an oncoming or in progress asthma attack. Examples of quick-relief medications include short-acting beta-2 agonists, such as albuterol, prednisone, methylprednisolone and hydrocortisone.
Allergy-desensitization shots (immunotherapy) can also be utilized, where a series of therapeutic injections containing small doses of those allergens. These injections are administered once a week for a few months, then once a month for a period of three to five years, the theory being that over time, the patient will lose their sensitivity to the allergens. Additionally, blocking the action of human immunoglobulin E (IgE), which is commonly involved with allergies when present in high amounts in the body, is still another route for treating asthma. Omalizumab (marketed under the name XOLAIR) is a monoclonal antibody made by Genentech/Novartis and used mainly in allergy-related asthma therapy, with the purpose of reducing allergic hypersensitivity. XOLAIR (omalizumab) is a recombinant DNA-derived humanized IgG1k monoclonal antibody that selectively binds to human immunoglobulin E (IgE), and limits the degree of release of mediators of the allergic response, and thus attenuates the asthmatic response.
Arthritis
Arthritis is a joint disorder that results in inflammation at an area of a patient where two different bones meet. As such, arthritis is typically accompanied by joint pain, that can be the result of wear and tear of cartilage (e.g. osteoarthritis) to pain associated with inflammation resulting from an overactive immune system (e.g. rheumatoid arthritis). Arthritis is classified as a rheumatic disease and as such affects joints, muscles, ligaments, cartilage, tendons, and may have the potential to affect internal body organs.
Rheumatoid arthritis (RA) is a long-term disease that causes inflammation of the joints and surrounding tissues and may affect other organs/tissues depending on the patient. RA is considered an autoimmune disease, and it appears to affected women more often than men. Joints of the extremities (i.e. arms and legs) are most commonly affected and including but limited to the wrists, fingers, knees, feet, and ankles.
Symptoms of arthritis include inflammation; pain and limited joint function e.g., joint stiffness, swelling, redness, and warmth. In persons suffering RA, symptoms in some patients can include fever, joint swelling, fatigue, and pain in various organs such as the lungs, heart, or kidneys.
Various treatment options are typically utilized to treat arthritis and include NSAIDs (nonsteroidal anti-inflammatory drugs), COX-2 inhibitors, various analgesics and corticosteroids. In some instances, a physician may choose to directly inject a medicament into the affected joint. This is known as viscosupplementation, and involves injection of gel-like substances (hyaluronates) into the subject a joint to supplement the viscous properties of synovial fluid in the joint. For example, SYNVISC® is an FDA-approved elastic and viscous substance made from hyaluronan that is injected into the knee to provide pain relief from osteoarthritis.
Clostridial Toxins
The genus Clostridium has more than one hundred and twenty seven species, grouped according to their morphology and functions. The anaerobic, gram positive bacterium Clostridium botulinum produces a potent polypeptide neurotoxin, botulinum toxin, which causes a neuroparalytic illness in humans and animals referred to as botulism. The spores of Clostridium botulinum are found in soil and can grow in improperly sterilized and sealed food containers of home based canneries, which are the cause of many of the cases of botulism. The effects of botulism typically appear 18 to 36 hours after eating the foodstuffs infected with a Clostridium botulinum culture or spores. The botulinum toxin can apparently pass unattenuated through the lining of the gut and attack peripheral motor neurons. Symptoms of botulinum toxin intoxication can progress from difficulty walking, swallowing, and speaking to paralysis of the respiratory muscles and death.
About 50 picograms of a commercially available botulinum toxin type A (a purified neurotoxin complex available from Allergan, Inc., of Irvine, Calif. under the tradename BOTOX® in 100 unit vials) is a LD50 in mice (i.e. 1 unit). One unit of BOTOX® contains about 50 picograms (about 56 attomoles) of botulinum toxin type A complex. Interestingly, on a molar basis, botulinum toxin type A is about 1.8 billion times more lethal than diphtheria, about 600 million times more lethal than sodium cyanide, about 30 million times more lethal than cobra toxin and about 1 2 million times more lethal than cholera. Singh, Critical Aspects of Bacteria/Protein Toxins, pages 63-84 (chapter 4) of Natural Toxins II, edited by B. R. Singh et al., Plenum Press, New York (1976) (where the stated LD50 of botulinum toxin type A of 0.3 ng equals 1 unit is corrected for the fact that about 0.05 ng of BOTOX® equals 1 unit). One unit (U) of botulinum toxin is defined as the LD50 upon intraperitoneal injection into female Swiss Webster mice weighing 18 to 20 grams each.
Seven immunologically distinct botulinum neurotoxins have been characterized, these being respectively botulinum neurotoxin serotypes A, B, C1, D, E, F and G, each of which is distinguished by neutralization with type-specific antibodies. The different serotypes of botulinum toxin vary in the animal species that they affect and in the severity and duration of the paralysis they evoke. For example, it has been determined that botulinum toxin type A is 500 times more potent, as measured by the rate of paralysis produced in the rat, than is botulinum toxin type B. Additionally, botulinum toxin type B has been determined to be non-toxic in primates at a dose of 480 U/kg which is about 12 times the primate LD50 for botulinum toxin type A. Moyer E et al., Botulinum Toxin Type 8: Experimental and Clinical Experience, being chapter 6, pages 71-85 of “Therapy With Botulinum Toxin,” edited by Jankovic, J. et al. (1994), Marcel Dekker, Inc. Botulinum toxin apparently binds with high affinity to cholinergic motor neurons, is translocated into the neuron, and blocks the release of acetylcholine. Additional uptake can take place through low affinity receptors, as well as by phagocytosis and pinocytosis.
Regardless of stereotype, the molecular mechanism of toxin intoxication appears to be similar and to involve at least three steps or stages. In the first step of the process, the toxin binds to the presynaptic membrane of the target neuron through a specific interaction between the heavy chain, H chain, and a cell surface receptor; the receptor is thought to be different for each type of botulinum toxin and for tetanus toxin. The carboxyl end segment of the H chain, HC, appears to be important for targeting of the toxin to the cell surface. In the second step, the toxin crosses the plasma membrane of the poisoned cell. The toxin is first engulfed by the cell through receptor-mediated endocytosis, and an endosome containing the toxin is formed. The toxin then escapes the endosome into the cytoplasm of the cell. This step is thought to be mediated by the amino end segment of the H chain, HN, which triggers a conformational change of the toxin in response to a pH of about 5.5 or lower. Endosomes are known to possess a proton pump which decreases intra-endosomal pH. The conformational shift exposes hydrophobic residues in the toxin, which permits the toxin to embed itself in the endosomal membrane. The toxin (or at a minimum the light chain) then translocates through the endosomal membrane into the cytoplasm.
The last step of the mechanism of botulinum toxin activity appears to involve reduction of the disulfide bond joining the heavy chain, H chain, and the light chain, L chain. The entire toxic activity of botulinum and tetanus toxins is contained in the L chain of the holotoxin; the L chain is a zinc (Zn2+) endopeptidase which selectively cleaves proteins essential for recognition and docking of neurotransmitter-containing vesicles with the cytoplasmic surface of the plasma membrane, and fusion of the vesicles with the plasma membrane. Tetanus neurotoxin, botulinum toxin types B, D, F, and G, cause degradation of synaptobrevin (also called vesicle-associated membrane protein (VAMP)), a synaptosomal membrane protein. Most of the VAMP present at the cytoplasmic surface of the synaptic vesicle is removed as a result of any one of these cleavage events. Botulinum toxin serotype A and E cleave SNAP-25. Botulinum toxin serotype C1 was originally thought to cleave syntaxin, but was found to cleave syntaxin and SNAP-25. Each of the botulinum toxins specifically cleaves a different bond, except botulinum toxin type B (and tetanus toxin) which cleave the same bond. Each of these cleavages block the process of vesicle-membrane docking, thereby preventing exocytosis of vesicle content.
Botulinum toxins have been used in clinical settings for the treatment of neuromuscular disorders characterized by hyperactive skeletal muscles (i.e. motor disorders). Almost twenty years ago, in 1989, a botulinum toxin type A complex was approved by the U.S. Food and Drug Administration for the treatment of blepharospasm, strabismus and hemifacial spasm. Subsequently, a botulinum toxin type A was also approved by the FDA for the treatment of cervical dystonia and for the treatment of glabellar lines, and a botulinum toxin type B was approved for the treatment of cervical dystonia. Non-type A botulinum toxin serotypes apparently have a lower potency and/or a shorter duration of activity as compared to botulinum toxin type A. Clinical effects of peripheral intramuscular botulinum toxin type A are usually seen within one week of injection. The typical duration of symptomatic relief from a single intramuscular injection of botulinum toxin type A averages about three months, although significantly longer periods of therapeutic activity have been reported.
Although all the botulinum toxin serotypes apparently inhibit release of the neurotransmitter acetylcholine at the neuromuscular junction, they do so by affecting different neurosecretory proteins and/or cleaving these proteins at different sites. For example, botulinum types A and E both cleave the 25 kiloDalton (kD) synaptosomal associated protein (SNAP-25), but they target different amino acid sequences within this protein. Botulinum toxin types B, D, F and G act on vesicle-associated protein (VAMP, also called synaptobrevin), with each serotype cleaving the protein at a different site. Finally, botulinum toxin type C1 has been shown to cleave both syntaxin and SNAP-25. These differences in mechanism of action may affect the relative potency and/or duration of action of the various botulinum toxin serotypes. Apparently, a substrate for a botulinum toxin can be found in a variety of different cell types. See e.g. Biochem J 1; 339 (pt 1):159-65.1999, and MovDisord, 10(3):376:1995 (pancreatic islet B cells contains at least SNAP-25 and synaptobrevin).
The molecular weight of the botulinum toxin protein molecule, for all seven of the known botulinum toxin serotypes, is about 150 kD. Interestingly, the botulinum toxins are released by Clostridial bacterium as complexes comprising the 150 kD botulinum toxin protein molecule along with associated non-toxin proteins. Thus, the botulinum toxin type A complex can be produced by Clostridial bacterium as 900 kD, 500 kD and 300 kD forms. Botulinum toxin types B and C1 are apparently produced as only a 700 kD or 500 kD complex. Botulinum toxin type D is produced as both 300 kD and 500 kD complexes. Finally, botulinum toxin types E and F are produced as only approximately 300 kD complexes. The complexes (i.e. molecular weight greater than about 150 kD) are believed to contain a non-toxin hemaglutinin protein and a non-toxin and non-toxic nonhemaglutinin protein. These two non-toxin proteins (which along with the botulinum toxin molecule comprise the relevant neurotoxin complex) may act to provide stability against denaturation to the botulinum toxin molecule, and protection against digestive acids when toxin is ingested. Additionally, it is possible that the larger (greater than about 150 kD molecular weight) botulinum toxin complexes may result in a slower rate of diffusion of the botulinum toxin away from a site of intramuscular injection of a botulinum toxin complex.
In vitro studies have indicated that botulinum toxin inhibits potassium cation induced release of both acetylcholine and norepinephrine from primary cell cultures of brainstem tissue. Additionally, it has been reported that botulinum toxin inhibits the evoked release of both glycine and glutamate in primary cultures of spinal cord neurons and that in brain synaptosome preparations botulinum toxin inhibits the release of each of the neurotransmitters acetylcholine, dopamine, norepinephrine (Habermann E., et al., Tetanus Toxin and Botulinum A and C Neurotoxins Inhibit Noradrenaline Release From Cultured Mouse Brain J Neurochem 51(2); 522-527:1988)), CGRP, substance P, and glutamate (Sanchez-Prieto, J., et al., Botulinum Toxin A Blocks Glutamate Exocytosis From Guinea Pig Cerebral Cortical Synaptosomes, Eur J. Biochem 165; 675-681:1897). Thus, when adequate concentrations are used, stimulus-evoked release of most neurotransmitters is blocked by botulinum toxin. See e.g. Pearce, L. B., Pharmacologic Characterization of Botulinum Toxin For Basic Science and Medicine, Toxicon 35(9); 1 373-1 412 at 1393; Bigalke H., et al., Botulinum A Neurotoxin Inhibits Non-Cholinergic Synaptic Transmission in Mouse Spinal Cord Neurons in Culture, Brain Research 360; 318-324:1985; Habermann E., Inhibition by Tetanus and Botulinum A Toxin of the release of [3H] Noradrenaline and [3H]GABA From Rat Brain Homogenate, Experientia 44; 224-226: 1988, Bigalke H., et al., Tetanus Toxin and Botulinum A Toxin Inhibit Release and Uptake of Various Transmitters, as Studied with Particulate Preparations From Rat Brain and Spinal Cord, Naunyn-Schmiedeberg's Arch Pharmacol 31 6; 244-251:1 981, and; Jankovic J. et al., Therapy With Botulinum Toxin, Marcel Dekker, Inc., (1994), page 5.
Botulinum toxin type A can be obtained by establishing and growing cultures of Clostridium botulinum in a fermenter and then harvesting and purifying the fermented mixture in accordance with known procedures. All the botulinum toxin serotypes are initially synthesized as inactive single chain proteins which must be cleaved or nicked by proteases to become neuroactive. The bacterial strains that make botulinum toxin serotypes A and G possess endogenous proteases and serotypes A and G can therefore be recovered from bacterial cultures in predominantly their active form. In contrast, botulinum toxin serotypes C1, D and E are synthesized by nonproteolytic strains and are therefore typically unactivated when recovered from culture. Serotypes B and F are produced by both proteolytic and nonproteolytic strains and therefore can be recovered in either the active or inactive form. However, even the proteolytic strains that produce, for example, the botulinum toxin type B serotype, only cleave a portion of the toxin produced. The exact proportion of nicked to unnicked molecules depends on the length of incubation and the temperature of the culture. Therefore, a certain percentage of any preparation of, for example, the botulinum toxin type B toxin, is likely to be inactive, possibly accounting for the known significantly lower potency of botulinum toxin type B, as compared to botulinum toxin type A (and thus the routine use of many thousands of units of botulinum toxin type B, as known in the art, see e.g. “Long-term safety, efficacy, and dosing of botulinum toxin type B (MYOBLOC®) in cervical dystonia (CD) and other movement disorders” Kumar R and Seeberger L C. Mov Disord 2002; 17(Suppl 5):S292-S293). The presence of inactive botulinum toxin molecules in a clinical preparation will contribute to the overall protein load of the preparation, which has been linked to increased antigenicity, without contributing to its clinical efficacy. Additionally, it is known that botulinum toxin type B has, upon intramuscular injection, a shorter duration of activity and is also less potent than botulinum toxin type A at the same dose level.
High quality crystalline botulinum toxin type A can be produced from the Hall A strain of Clostridium botulinum with characteristics of >3×107 U/mg, an A260/A278 of less than 0.60 and a distinct pattern of banding on gel electrophoresis. The known Schantz process can be used to obtain crystalline botulinum toxin type A, as set forth in Schantz, E. J., et al, Properties and use of Botuilnum toxin and Other Microbial Neurotoxins in Medicine, Microbiol Rev. 56; 80-99:1992. Generally, the botulinum toxin type A complex can be isolated and purified from an anaerobic fermentation by cultivating Clostridium botulinum type A in a suitable medium. The known process can also be used, upon separation out of the non-toxin proteins, to obtain pure botulinum toxins, such as for example: purified botulinum toxin type A with an approximately 150 kD molecular weight with a specific potency of 1-2×108 LD50 U/mg or greater; purified botulinum toxin type B with an approximately 156 kD molecular weight with a specific potency of 1-2×108 LD50 U/mg or greater; and purified botulinum toxin type F with an approximately 155 kD molecular weight with a specific potency of 1-2×107 LD50 U/mg or greater.
Botulinum toxins and/or botulinum toxin complexes can be obtained from List Biological Laboratories, Inc., Campbell, Calif.; the Centre for Applied Microbiology and Research, Porton Down, U.K.; Wako (Osaka, Japan), Metabiologics (Madison, Wis.) as well as from Sigma Chemicals of St Louis, Mo. Pure botulinum toxin can also be used to prepare a pharmaceutical composition for use in accordance with the present disclosure.
As with enzymes generally, the biological activities of botulinum toxins (which are intracellular peptidases) is dependant, at least in part, upon their 3-dimensional conformation. Thus, botulinum toxin type A is detoxified by heat, various chemicals, surface stretching, and surface drying. Additionally, it is known that dilution of the toxin complex obtained by the known culturing, fermentation and purification to the much lower toxin concentrations used for pharmaceutical composition formulation results in rapid detoxification of the toxin unless a suitable stabilizing agent is present. Dilution of the toxin from milligram quantities to a solution containing nanograms per milliliter presents significant difficulties because of the rapid loss of specific toxicity upon such great dilution. Since the toxin may be used months or years after the toxin containing pharmaceutical composition is formulated, the toxin can be stabilized with a stabilizing agent such as albumin and gelatin.
A commercially available botulinum toxin containing pharmaceutical composition is sold under the trademark BOTOX® (available from Allergan, Inc., of Irvine, Calif.). BOTOX® consists of a purified botulinum toxin type A complex, albumin and sodium chloride packaged in sterile, vacuum-dried form. Botulinum toxin type A is made from a culture of the Hall strain of Clostridium botulinum grown in a medium containing N-Z amine and yeast extract. The botulinum toxin type A complex is purified from the culture solution by a series of acid precipitations to a crystalline complex consisting of the active high molecular weight toxin protein and an associated hemagglutinin protein. The crystalline complex is re-dissolved in a solution containing saline and albumin and sterile filtered (0.2 microns) prior to vacuum-drying. The vacuum-dried product is stored in a freezer at or below −5° C. BOTOX® can be reconstituted with sterile, non-preserved saline prior to intramuscular injection. Each vial of BOTOX® contains about 100 U of Clostridium botulinum toxin type A purified neurotoxin complex, 0.5 milligrams of human serum albumin and 0.9 milligrams of sodium chloride in a sterile, vacuum-dried form without a preservative.
To reconstitute vacuum-dried BOTOX®, sterile normal saline without a preservative (0.9% Sodium Chloride Injection) is used by drawing up the proper amount of diluent in the appropriate size syringe. Since BOTOX® may be denatured by bubbling or similar violent agitation, the diluent is gently injected into the vial. For sterility reasons BOTOX® is preferably administered within four hours after the vial is removed from the freezer and reconstituted. During these four hours, reconstituted BOTOX® can be stored in a refrigerator at about 2° C. to about 8° C. Reconstituted, refrigerated BOTOX® has been reported to retain its potency for at least about two weeks (Neurology, 48:249-53, 1997). It has been reported that botulinum toxin type A has been used in clinical settings as follows:    (1) about 75-125 U of BOTOX® per intramuscular injection (multiple muscles) to treat cervical dystonia;    (2) 5-10 U of BOTOX® per intramuscular injection to treat glabellar lines (brow furrows) (5 units injected intramuscularly into the procerus muscle and 10 units injected intramuscularly into each corrugator supercilii muscle);    (3) about 30-80 U of BOTOX® to treat constipation by intrasphincter injection of the puborectalis muscle;    (4) about 1-5 Upper muscle of intramuscularly injected BOTOX® to treat blepharospasm by injecting the lateral pre-tarsal orbicularis oculi muscle of the upper lid and the lateral pre-tarsal orbicularis oculi of the lower lid;    (5) to treat strabismus, extraocular muscles have been injected intramuscularly with between about 1-5 U of BOTOX®, the amount injected varying based upon both the size of the muscle to be injected and the extent of muscle paralysis desired (i.e. amount of diopter correction desired);    (6) to treat upper limb spasticity following stroke by intramuscular injections of BOTOX® into five different upper limb flexor muscles, as follows:    (a) flexor digitorum profundus: 7.5 U to 30 U    (b) flexor digitorum sublimus: 7.5 U to 30 U    (c) flexor carpi ulnaris: 10 U to 40 U    (d) flexor carpi radialis: 15 U to 60 U    (e) biceps brachii: 50 U to 200 U. Each of the five indicated muscles has been injected at the same treatment session, so that the patient receives from 90 U to 360 U of upper limb flexor muscle BOTOX® by intramuscular injection at each treatment session;(7) to treat migraine, pericranial (injected symmetrically into glabellar, frontalis and temporalis muscles) injection of 25 U of BOTOX® has showed significant benefit as a prophylactic treatment of migraine compared to vehicle as measured by decreased measures of migraine frequency, maximal severity, associated vomiting and acute medication use over the three month period following the 25 U injection.
It is known that botulinum toxin type A can have an efficacy for up to 12 months (European J. Neurology 6 (Supp 4): S11-S1 150: 1999), and in some circumstances for as long as 27 months, when used to treat glands, such as in the treatment of hype rhydrosis. See e.g. Bushara K., Botulinum toxin and rhinorrhea, Otolaryngol Head Neck Surg 1996; 114(3):507, and The Laryngoscope 109:1344-1346:1999. However, the usual duration of effect of an intramuscular injection of BOTOX® is typically about 3 to 4 months.
The success of botulinum toxin type A to treat a variety of clinical conditions has led to interest in other botulinum toxin serotypes. Two commercially available botulinum type A preparations for use in humans are BOTOX® available from Allergan, Inc., of Irvine, Calif., and DYSPORT® available from Beaufour Ipsen, Porton Down, England. A botulinum toxin type B preparation (MYOBLOC®) is available from Solstice Pharmaceuticals of San Francisco, Calif.
A botulinum toxin has also been proposed for or has been used to treat otitis media of the ear (U.S. Pat. No. 5,766,605), inner ear disorders (U.S. Pat. Nos. 6,265,379; 6,358,926), tension headache, (U.S. Pat. No. 6,458,365), migraine headache pain (U.S. Pat. No. 5,714,468), post-operative pain and visceral pain (U.S. Pat. No. 6,464,986), hair growth and hair retention (U.S. Pat. No. 6,299,893), psoriasis and dermatitis (U.S. Pat. No. 5,670,484), injured muscles (U.S. Pat. No. 6,423,319) various cancers (U.S. Pat. Nos. 6,139,845), smooth muscle disorders (U.S. Pat. No. 5,437,291), and neurogenic inflammation (U.S. Pat. No. 6,063,768). Controlled release toxin implants are known (see e.g. U.S. Pat. Nos. 6,306,423 and 6,312,708) as is transdermal botulinum toxin administration (U.S. patent application Ser. No. 10/194,805). U.S. Patent Application Publication 2007/0048334 A1, Ser. No. 11/211,311 and filed Aug. 24, 2005, discloses the use of a botulinum toxin to improve gastric emptying and/or treating gastroesophageal reflux disease (GERD) by administration to a patient's head, neck and shoulder muscles. It is known that a botulinum toxin can be used to weaken the chewing or biting muscle of the mouth so that self inflicted wounds and resulting ulcers can heal (Payne M., et al, Botulinum toxin as a novel treatment for self mutilation in Lesch-Nyhan syndrome, Ann Neurol 2002 September; 52(3 Supp 1):S157).). U.S. Patent Application Publication 20050191321 A1, Ser. No. 11/039,506 and filed Jan. 18, 2004, discloses treating medication overuse disorders (MOD), by local administration of a Clostridial toxin. U.S. Patent Application Publication 20050147626 A1, Ser. No. 10/964,898 and filed Oct. 12, 2004 discloses treating or preventing, by peripheral administration of a botulinum toxin to or to the vicinity of a trigeminal sensory nerve, a neurological disorder and/or a neuropsychiatric disorder.
Additionally, a botulinum toxin may have an effect to reduce induced inflammatory pain in a rat formalin model. Aoki K., et al, Mechanisms of the antinociceptive effect of subcutaneous BOTOX®: Inhibition of peripheral and central nociceptive processing, Cephalalgia 2003 September; 23(7):649. Furthermore, it has been reported that botulinum toxin nerve blockage can cause a reduction of epidermal thickness. Li Y, et al., Sensory and motor denervation influences epidermal thickness in rat foot glabrous skin, Exp Neurol 1997; 147:452-462 (see page 459). U.S. Patent Application Publication 20050266029 A1, Ser. No. 11/159569 and filed on Jun. 22, 2005 relates to methods for treating pain associated with arthritis. Finally, it is known to administer a botulinum toxin to the foot to treat excessive foot sweating (Katsambas A., et al., Cutaneous diseases of the foot: Unapproved treatments, Clin Dermatol 2002 November-December; 20(6):689-699; Sevim, S., et al., Botulinum toxin-A therapy for palmar and plantar hyperhidrosis, Acta Neurol Belg 2002 December; 102(4):167-70), spastic toes (Suputtitada, A., Local botulinum toxin type A injections in the treatment of spastic toes, Am J Phys Med Rehabil 2002 October; 81(10):770-5), idiopathic toe walking (Tacks, L., et al., Idiopathic toe walking: Treatment with botulinum toxin A injection, Dev Med Child Neurol 2002; 44(Suppl 91):6), and foot dystonia (Rogers J., et al., Injections of botulinum toxin A in foot dystonia, Neurology 1993 April; 43(4 Suppl 2)). Tetanus toxin, as wells as derivatives (i.e. with a non-native targeting moiety), fragments, hybrids and chimeras thereof can also have therapeutic utility. The tetanus toxin bears many similarities to the botulinum toxins. Thus, both the tetanus toxin and the botulinum toxins are polypeptides made by closely related species of Clostridium (Clostridium tetani and Clostridium botulinum, respectively).
Additionally, both the tetanus toxin and the botulinum toxins are dichain proteins composed of a light chain (molecular weight about 50 kD) covalently bound by a single disulfide bond to a heavy chain (molecular weight about 100 kD). Hence, the molecular weight of tetanus toxin and of each of the seven botulinum toxins (non-complexed) is about 150 kD. Furthermore, for both the tetanus toxin and the botulinum toxins, the light chain bears the domain which exhibits intracellular biological (protease) activity, while the heavy chain comprises the receptor binding (immunogenic) and cell membrane translocational domains.
Additionally, both the tetanus toxin and the botulinum toxins exhibit a high, specific affinity for gangliocide receptors on the surface of presynaptic cholinergic neurons. Receptor mediated endocytosis of tetanus toxin by peripheral cholinergic neurons results in retrograde axonal transport, blocking of the release of inhibitory neurotransmitters from central synapses and a spastic paralysis. Contrarily, receptor mediated endocytosis of botulinum toxin by peripheral cholinergic neurons results in little if any retrograde transport, inhibition of acetylcholine exocytosis from the intoxicated peripheral motor neurons and a flaccid paralysis.
Finally, the tetanus toxin and the botulinum toxins resemble each other in both biosynthesis and molecular architecture. Thus, there is an overall 34% identity between the protein sequences of tetanus toxin and botulinum toxin type A, and a sequence identity as high as 62% for some functional domains. Binz T. et al., The Complete Sequence of Botulinum Neurotoxin Type A and Comparison with Other Clostridial Neurotoxins, J Biological Chemistry 265(16); 9153-9158:1990.
Acetylcholine
Typically only a single type of small molecule neurotransmitter is released by each type of neuron in the mammalian nervous system. The neurotransmitter acetylcholine is secreted by neurons in many areas of the brain, but specifically by the large pyramidal cells of the motor cortex, by several different neurons in the basal ganglia, by the motor neurons that innervate the skeletal muscles, by the preganglionic neurons of the autonomic nervous system (both sympathetic and parasympathetic), by the postganglionic neurons of the parasympathetic nervous system, and by some of the postganglionic neurons of the sympathetic nervous system. Essentially, only the postganglionic sympathetic nerve fibers to the sweat glands, the piloerector muscles and a few blood vessels are cholinergic as most of the postganglionic neurons of the sympathetic nervous system secret the neurotransmitter norepinephine. In most instances acetylcholine has an excitatory effect. However, acetylcholine is known to have inhibitory effects at some of the peripheral parasympathetic nerve endings, such as inhibition of heart rate by the vagal nerve.
The efferent signals of the autonomic nervous system are transmitted to the body through either the sympathetic nervous system or the parasympathetic nervous system. The preganglionic neurons of the sympathetic nervous system extend from preganglionic sympathetic neuron cell bodies located in the intermediolateral horn of the spinal cord. The preganglionic sympathetic nerve fibers, extending from the cell body, synapse with postganglionic neurons located in either a paravertebral sympathetic ganglion or in a prevertebral ganglion. Since, the preganglionic neurons of both the sympathetic and parasympathetic nervous system are cholinergic, application of acetylcholine to the ganglia will excite both sympathetic and parasympathetic postganglionic neurons.
Acetylcholine activates two types of receptors, muscarinic and nicotinic receptors. The muscarinic receptors are found in all effector cells stimulated by the postganglionic neurons of the parasympathetic nervous system, as well as in those stimulated by the postganglionic cholinergic neurons of the sympathetic nervous system. The nicotinic receptors are found in the synapses between the preganglionic and postganglionic neurons of both the sympathetic and parasympathetic. The nicotinic receptors are also present in many membranes of skeletal muscle fibers at the neuromuscular junction.
Acetylcholine is released from cholinergic neurons when small, clear, intracellular vesicles fuse with the presynaptic neuronal cell membrane. A wide variety of non-neuronal secretory cells, such as, adrenal medulla (as well as the PC12 cell line) and pancreatic islet cells release catecholamines and parathyroid hormone, respectively, from large dense-core vesicles. The PC12 cell line is a clone of rat pheochromocytoma cells extensively used as a tissue culture model for studies of sympathoadrenal development. Botulinum toxin inhibits the release of both types of compounds from both types of cells in vitro, permeabilized (as by electroporation) or by direct injection of the toxin into the denervated cell. Botulinum toxin is also known to block release of the neurotransmitter glutamate from cortical synaptosomes cell cultures.
What is needed therefore are effective and efficient methods for treating arthritis, respiratory disorders, such as COPD, asthma, bronchitis and for treating/alleviating (e.g. lowering) coronary risk factors, such as hypertension, high cholesterol, high triglyceride levels, diabetes, hyperlipidemia, and obesity that do not require daily administration and/or strict patient compliance.